Decreased contractility is a hallmark of gallbladder disorders, and it is associated with inflammation and gallstone formation. Recent reports suggest that decreased gallbladder motility is caused by elevated cholesterol and hydrophobic salts in the bile, and that ensuing inflammation and impairments in gallbladder emptying lead to stone formation. Furthermore, mounting evidence suggests that hydrophilic bile salts may have therapeutic potential by restoring gallbladder smooth muscle (GBSM) contractility and reducing gallstone development and inflammation. Nevertheless, the interrelationships between decreased contractility, inflammation, and gallstone formation are not understood, and this reflects our lack of understanding of the cellular events that lead to decreased resting tone and postprandial contractions in gallbladder disease. This competing renewal will address these fundamental gaps in our knowledge. The overall objectives of this grant application are to: (1) elucidate the cellular and ionic mechanisms by which GBSM contractility is disrupted in gallstone disease, (2) resolve the mechanism by which hydrophobic bile salts interact with GBSM;(3) elucidate the role of inflammation in smooth muscle dysfunction and associated biliary stasis in gallstone disease;and (4) elucidate the role of hydrophilic bile salts in the protection of gallbladder function by preventing or reversing these disruptions. Two experimental models will be used. In one model, we will use wild type and transgenic mice fed a lithogenic (gallstone forming) diet to evaluate the functional changes that occur in GBSM during the progression of gallstone disease in normal mice, mice that do not develop cholecystitis, and mice lacking nuclear or membrane bile acid receptors. We will also evaluate GBSM function in mice fed a lithogenic diet while being treated with the protective hydrophilic bile salt, ursodeoxycholic acid, or a cyclooxygenase inhobitor. The second model involves evaluation of the effects of cholesterol and hydrophobic bile salts applied in vitro to GBSM preparations, with or without prior application of ursodeoxycholic acid. This approach allows us to examine the actions of these compounds individually on GBSM in a controlled environment. This grant proposal involves an integrated approach using state-of-the-art techniques to investigate gallbladder pathophysiology from single molecules to the intact tissue during the progression of disease in the animal. Techniques to be used include Ca2+ imaging of GBSM cells in intact muscle bundles, intracellular and patch clamp recording from intact and isolated GBSM cells, respectively, and muscularis tension measurements. Together with our previous studies on smooth muscle function, we will provide insights on how gallbladder motor function is disrupted leading to biliary stasis, and the relationship between disrupted motor function and inflammation. Furthermore, these studies will elucidate the therapeutic potential of hydrophilic bile salts on GBSM to prevent or reverse cellular changes that underlie decrease gallbladder contractility, and uncover the cellular mechanisms that mediate their actions. PUBLIC HEALTH RELEVANCE: Gallstone disease is an extremely common disorder that involves a decrease in the ability of the gallbladder to contract and empty bile into the intestine, thus leading to gallstone formation. The objectives of this grant application are to understand the cellular mechanisms by gallbladder muscle is disrupted in gallstone disease, and to determine whether and how hydrophilic bile salts can protect the gallbladder from these changes.